Biocompatible polyorganosiloxane composition for cell culture apparatus

ABSTRACT

A polyorganosiloxane composition having a biocompatible surface thereon is disclosed. The biocompatible surface results from the derivatization, or amination, of the surface intended for cell contact. More specifically, the present invention is a polyorganosiloxane composition in which the surface is either treated with a primary amine and optional peptide or the surface is co-cured with a primary amine-containing silane or siloxane. The aminated polyorganosiloxane has utility as a cell culture substrate or in a variety of artificial organ applicatons such as breast implants, synthetic blood vessels, joints, tendons and heart valves. A vacuum apparatus for use with specialized cell cultrue plates incorporating the biocompatible polyorganosiloxane composition is also disclosed.

This application is a division of application Ser. No. 046,440, filedMay 4, 1987, now U.S. Pat. No. 4,789,601.

FIELD OF THE INVENTION

The present invention relates to surface-modified polyorganosiloxanecompositions which demonstrate improved biocompatibility both in vitroand in vivo, and to a cell culture apparatus incorporating suchcompositions.

BACKGROUND OF THE INVENTION

Although the use of synthetic polymers in technology and in everydaylife is widespread, the use of polymers in clinical and laboratorymedicine has been cautious and limited. This restricted use is unrelatedto need; suitable synthetic polymers are increasingly in demand for usein the fabrication of artificial organs and membranes for hemodialysisor oxygenation, in the preparation of plasma or blood substitutes, andin the manufacture of implanted or soluble polymers as substrates forthe slow release of drugs, hormones or other physiologically-activeagents.

Unfortunately, even those synthetic polymers which demonstraterelatively low cytotoxicity, such as the various silicone resins,typically demonstrate at least some degree of bioincompatibility. Forexample, a silicone resin implant embedded within mammalian or humantissue ordinarily eventuates encapsulation of that implant, includingepithelial encapsulation or thickening and/or keratinization of thesurrounding connective tissues. A similar phenomenon in vitro preventscells from adhering to many synthetic polymer substrates when thosesubstrates are subjected to elongation or other stresses.

With respect to in vitro cell cultures, specifically, there is as greata need for elastomeric substrates to which cells can adhere in vitro asthere is a need for biocompatible polymers for in vivo applications.This need arose from developments in the area of in vitro flexing ofcell cultures, which fiexing techniques off certain advantages overconventional cell culture methods.

Conventional culture plates or bottles used for the propagation of cellsin vitro are typically manufactured from polystyrene or glass. Therouting method for cuituring cells includes inoculating the cells intoflasks, single culture dishes or multi-well plates, adding a nutrientmedium and incubating the cells under controlled conditions. Alternativemethods for the in vitro culturing of cells include growing cells incontinuously rolling glass or plastic bottles, so that the cells adhereto the wall of a culture vessel beneath continually rotated medium(cells may alternately be grown in fluted roller bottles that haveincreased inside surface area), or culturing cells on glass or complexpolysaccharide beads, tissue segments or in suspension in a suitableculture medium. With all these methods, however, the culture medium doesnot exert any deforming stress upon the cells themselves such as wouldsimulate the in vivo stresses applied by tendons, for example, or thecyclic stresses exerted by the heart or lungs on their constituentcells.

To simulate what cells experience in the way of physical deformation inthe environment of the lung, cells can be adhered to and grown upon anelastomeric substrate which is cyclically stretched to 20 percentelongation, fifteen times a minute, in order to simulate a restingsituation. Lung cells may also be cyclically stretched at 20 percentelongation, 40 times a minute, to simulate an exercise period. Suchresearch may be tailored to address such questions as whether cells aremore susceptible to viral infection when they are cyclically stretchedor at rest, or whether macrophages phagocytose bacteria more readily ifthey are subjected to cyclic deformation, and related questions. Theanswers to these questions can then be considered inthe development oftreatment plans for patients having viral or bacterial infections.

One system for the in vitro flexing of cells in culture is documented inBanes, A.J. et al., "A New Vacuum-Operated Stress-Providing InstrumentThat Applies Static or Variable Duration Cyclic Tension or Compressionto Cells In Vitro," J. Cell Sci., 1985. In that published protocol,however, physical limitations of the plastic (polystyrene) Petri dishprecluded more than a limited amount of cyclic deformation in the cellsubstrate (Petri dish base). (Related in vitro systems are documented inSomjen, D. et al., "Bone Remodelling Induced by Physical Stress inProstaglandin E₂ Mediated," Biochimica et Biophysica Acta, 627 (1980)91-100; Leung, D. Y. M. et al., "A New In Vitro System for Studying CellResponse to Mechanical Stimulation," Experimental Cell Research, 109(1977) 285-298; Leung, D. Y. M. et al., "Cyclic Stretching StimulatesSynthesis of Matrix Components by Arterial Smooth Muscle Cells InVitro," Science, 191 (1976) 475-477; Hasagawa et al. "MechanicalStretching Increases the Number of Cultured Bone Cells Synthesizing DNAand Alters Their Pattern of Protein Synthesis," Calcif Tissue Int, 37(1985) 431-436; and Brunette, D.M. et al., "Mechanical StretchingIncreases the Number of Epithelial Cells Synthesizing DNA in Culture,"J. Cell Sci, 69 (1984) 35-45.

In view of all of the above, a need remains for a low cytotoxicitysynthetic polymer composition which does not promote encapsulation invivo or cellular nonadherence in vitro. Ideally, such a compositionwould also offer the various benefits of the silicone resin compositionswhich are generally known to demonstrate both low cytotoxicity and hightensile and flexural strength.

SUMMARY OF THE INVENTION

In order to meet this need, the present invention is apolyorganosiloxane composition having a biocompatible surface thereon.The biocompatible surface results from the derivatization of the surfaceintended for cell contact. More specifically, the present invention is apolyorganosiloxane composition in which the surface is embedded withcarbon particles, or is treated with a primary amine and optionalpeptide, or is co-cured with a primary amine-or carboxyl-containingsilane or siloxane. The derivatized polyorganosiloxane has utility as acell culture substrate or in a variety of artificial organ applicationssuch as breast implants, synthetic blood vessels, joints, tendons, heartvalves and the like. A vacuum apparatus for use with specialized cellculture plates incorporating the biocompatible polyorganosiloxanecomposition is also disclosed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an exploded perspective view of a six-well culture platecontaining a biocompatible polyorganosiloxane composition according tothe present invention and a cover therefore;

FIG. 2 is plan view of the culture plate illustrated in FIG. 1;

FIG. 3 is a section taken along lines III--III in FIG. 2;

FIG. 4 is an exploded perspective view of a vacuum apparatus suitablefor use in association with the culture plate shown in FIG. 1;

FIG. 5 is a perspective view of the vacuum apparatus of FIG. 4;

FIG. 6 is a section taken along lines VI--VI in FIG. 5; and

FIG. 7 is a schematic diagram of an apparatus for regulating the vacuumapparatus of FIG. 3.

DETAILED DESCRIPTION OF THE INVENTION

The surface modification of the polyorganosiloxane compositionsaccording to the present invention is accomplished by one or more ofthree methods. The composition surface may be embedded with carbonparticles, may be treated with a primary amine and optional peptide, ormay be co-cured with a primary amine- or carboxyl-containing silane orsiloxane. It is believed that, when present, the amino or carboxylgroups and optional peptides orient to the surface of thepolyorganosiloxane composition and provide the biocompatible surface.the derivatization reaction may be carried out on the surface of eitheran uncured or a cured polyorganosiloxane. In every case, however,derivatization is conducted on the surface of the polyorganosiloxane.

A first method for the derivatization of a cured or uncuredpolyorganosiloxane surface, such as a membrane, comprises embedding thesurface with a plurality of elemental carbon particles. For example, theuncured surface may be suspended over a bunsen burner flame both todeposit fine elemental carbon particles on and to cure said surface. Theresulting surface demonstrates improved biocompatibility.

The second method for the derivatization of a cured polyorganosiloxanemembrane comprises an amination method which includes two basic steps.First, the membrane surface is treated for 30 minutes under ambientconditions, with swirling, with between 0.5 and 1 ml. 1 N. HCl for eachcm² of its surface area. The acid is then decanted. The surface is thencontacted, for 30 minutes and again under ambient conditions, withbetween 0.5 and 1 ml. 1 M. NH₄ OH per cm² surface. In the alternative,after the acid is decanted the surface may be exposed to ammonia vaporfor 15 minutes in a bell jar. The resultant modified surface is washedwith water and permitted to dry. Other acids and primary amines may besubstituted in stoichiometrically equivalent amounts, such as HFl, HBr(or other halide containing acids) NH₄ Cl or NH₄ HCO₃. It is believedthat the surface thus treated demonstrates biocompatibility due to thepresence of amino groups pendant from the treated polyorganosiloxane andoriented to the polymer surface.

A third method for treating polyorganosiloxane surfaces includes theamination treatment described above followed by an optional peptidation.After the acidification and amination steps, followed by water washing,the surface is treated by contacting it with between 0.5 and 1 ml. 1millimolar to 1 nanomolar glutaraldehyde per cm². Reactive equivalentamounts of other aldehydes, such as acetaldehyde or butyraldehyde may besubstituted. The glutaraldehyde-treated surface is then contacted withan aqueous peptide which typically has both amine and carboxylfunctionalities. Ordinarily, the peptide selected will have between 2and 40 amino acids in linear configuration so as to provide amine andcarboxyl functionality at opposing terminal ends of the peptide.However, larger peptides and proteins having molecular weights ofseveral thousand may also be used. A final water wash follows. It isbelieved that the aldehyde creates a Schiff's base in reaction with thebound amine, leaving a free aldehyde which then reacts with the aminogroup of the peptide. The resultant aminated/peptidatedpolyorganosiloxane therefore provides a biocompatible primaryamine-containing carboxyl-terminated surface, which biocompatibility isparticularly enhanced when the peptide is selected for itshistocompatibility with the specific cell culture. For any givenapplication, peptide compatibility may be determined by means known inthe art.

A fourth method of derivatization comprises the co-curing of apolyorganosiloxane with a primary amine- or carboxyl-containing silaneor siloxane. (The term "co-curing" signifies that at least one of theadjacent silane- or siloxane-containing compositions is cured in situ.)Exemplary compounds include 3-aminopropyltriethoxysilane,2-aminoethyltrimethoxysilane, trimethylsilylformic acid,3-(trichlorosilyl) butanoic acid, and 1,1,1-trichloro-N-(trimethylsilyl)silanamine. Suitable diluents for the primary amine- orcarboxyl-containing silane or siloxane include methoxy trimethylsilane,trimethoxysilane, chlorodimethylsilane and chlorotriisocyanatosilane.The silane or siloxane (with or without silane diluent) is applied inaqueous solution or in aqueous buffer solution at substantially neutralto basic pH to cover a cured polyorganosiloxane surface in entirety. Anyof the buffers ordinarily used in the preparation of cell culture mediaare suitable for use as the solvent or carrier for the silane orsiloxane such as, for example, the 20 mM HEPES buffer well known in theart. The resultant layers are co-cured at room temperature for a periodof not less than 15 minutes nor more than twenty-four hours. Curing maybe effected at elevated temperatures if desired. The primary amine- orcarboxyl-containing silane or siloxane may alternatively be coated ontothe uncured polyorganosiloxane, with the subsequent co-curing of the twolayers in the same manner as would have been chosen for the curing ofthe base polyorganosiloxane layer alone. The cured surfaces may befurther treated with the optional peptidation or carbon particleembedding treatment described above.

After preparation is complete by any one of the above methods, thecomposition is washed with water or buffer, sterilized such as withultraviolent light, and packaged for storage prior to use. Other meansof sterilization include microwave energy, gamma radiation and othersterilization means known in the art.

Specific derivatization methods are illustrated with particularity inthe Examples, infra.

Biocompatible derivatized polyorganosiloxanes in accordance with themethods described above have a vast number of potential applications,but one particularly important use for such elastomers is in the invitro flexing of cell culture substrates. Even as cell culturesubstrates, the use of the derivatized polyorganosiloxanes of thepresent invention are limitless: the compositions may be used alone, incombination with a wide variety of cell culture vessels, or in any otherapplication in which adherent cell culture growth is desired.

One embodiment of a cell culture plate 10, utilizing the biocompatiblepolyorganosiloxane compositions of the present invention, is shown inFIGS. 1-3. The culture plate 10 is a substantially flat, rectangularlyshaped structure including a flat upper surface 12, and side walls 13,14 and end walls 15, 16 extending downwardly from the outer edges of theupper surface 12 and joined together along adjacent edges. A pluralityof open topped wells 18 extend downwardly from the upper surface 12.While the culture plate 10 shown in FIGS. 1-3 includes six wells 18 in atwo by three array, it is to be understood that any number of wells 18,even as few as one, may be included as desired.

Each well 18 includes a cylindrically shaped side wall 20 and terminatesin a substantially flat well base 22 attached to side wall 20. The wellbase 22 includes an anchor ring 24 integral with the side wall 20. Theremainder of the well base 22 is formed by an elastomeric membrane 26which both covers the anchor ring 24 and fills the opening defined bythe anchor ring 24. The anchor ring 24 may include a plurality of holes28, preferably frustoconically shaped, extending therethrough. The holes28 are filled with elastomeric material continuous with the elastomericmembrane 26, which structure aids in securing the membrane 26 in placein the well base 22. The membrane 26 is fabricated from asurface-modified polyorganosiloxane composition as described above.

The wells 18 extend downwardly to an extend slightly below the loweredge of side walls 13, 14 and end walls 15, 16. The culture plate 10includes a protective base rim 30 which extends downwardly from thelower edges of side walls 13, 14 and end walls 15, 16 and beyond thebottom surface of the well bases 22. The base rim 30 functions toelevate, and to prevent any scratching of or damage to, the well bases22.

The upper surface 12 of the culture plate, and hence the plurality ofwells 18, may be closed off by a cover 32 or the like. The cornersbetween adjacent end walls and side walls may be configured to enableconsistent orientation of the culture plate. As shown in FIGS. 1 and 2,corner 34 between side wall 13 and end wall 15 and corner 35 betweenside wall 14 and end wall 15 are beveled. The cover 32 also hascorrespondingly beveled corners 36 and 37. The outer surface of the baserim 30 may be grooved to provide for more secure handling of the cultureplate 10.

The culture plate 10 shown in FIGS. 1-3 may be manufactured from acommercially available Falcon six-well culture plate formed of 1.5 mmthick polystyrene. The Falcon culture plate includes a well base formedof a solid layer of substantially planar polystyrene. The well base ofthe Falcon culture plate can be partially excised, leaving a base anchorring 24 of polystyrene in each well 18. A plurality of frustoconicalholes 28 are drilled into each anchor ring 24, with the narrow end ofthe hole at the upper surface of the anchor ring 24. A suitable releaselayer is affixed just beneath the anchor rings 24 by means known in theart, and an uncured polyorganosiloxane composition is deposited withineach well 18. One example of a suitable polyorganosiloxane compositionis the Dow-Corning MDX4-4210 (sold under the trademark SILASTIC®); anexemplary formulation is 85 parts Dow-Corning MDX4-4210, 15 parts of thematching lot number catalyst and 15 parts medical grade silicone oil.The uncured polyorganosiloxane which results from such or a similaradmixture is poured, in predetermined amounts, into each well 18 to forma membrane in the bottom thereof, and at the same time the uncuredcomposition flows into each of the frustoconical holes 28 in the anchorring 24. The polyorganosiloxane may then be deaerated, cured and therelease layer removed to yield the culture plate 10 of FIGS. 1-3.

Deaeration of, or removal of air bubbles from, the uncuredpolyorganosiloxane is particularly important in the preparation of anoptically clear well base 22. (Optical clarity facilitates microscopicexamination of the elastomer bearing cell culture, for example.) Airbubbles may be removed by means known in the art or may specifically beremoved by a specialized centrifuging technique. In order to deaerate bycentrifugation, the individual culture plates 10 with or without theircovers 32 are placed in centrifuge receptacles adapted to receive them.The plates are then subjected to centrifugation at 800-1200 timesgravity for 3-6 minutes. The plates 10 are removed from the centrifuge,are rotated or "rocked" by hand or machine to equilibrate thepolyorganosiloxane well base 22 to as close to a flat membrane aspossible, the plates are cured in an oven at approximately 60° C. for 45minutes and, upon removal from the oven and cooling, the release layeris removed. The upper surfaces of the well bases 22 of the culture plate10 may then be derivatized by any of the methods described above and asdescribed with particularity in the Examples, infra.

In general, the culture plate 10 in accordance with the presentinvention provide well bases 22 which, in addition to providing asubstrate to which cells can adhere, may be elongated or otherwisestressed by a number of means. One particularly convenient method forelongating an elastomeric cell substrate such as the well base 22includes selectively subjecting an enclosed area immediately beneath thewell base 22 to a controlled vacuum source. Such vacuum elongation mightbe accomplished by individual vacuum ports affixed beneath each wellbase 22. A simpler yet more effective apparatus for subjecting each wellbase 22 to selective vacuum is illustrated in the vacuum apparatus ofFIGS. 4-6.

The vacuum apparatus shown in FIGS. 4-6 includes a vacuum plenum 40formed from a solid, flat, rectangular sheet of Plexiglas.sup.™ or thelike. The upper surface of the plenum 40 has a plurality of vacuumchannels cut therein and having a depth less than the thickness of theplenum 40. A main vacuum channel 42 extends down the middle of theplenum 40 substantially along its entire length. A plurality of sidevacuum channels 44 extend perpendicularly from each side of the mainvacuum channel 42 and are in fluid communication therewith. A hole isdrilled through one end of the plenum 40 and is fitted with a nipple 46which is in fluid communication with the main vacuum channel 42. Thenipple 46 is adapted to receive a vacuum hose 48 which connects theplenum 40 to a source of vacuum (not shown).

Each vacuum channel 42, 44 is open to the upper surface of the plenum 40at every point along its length. In order to close off the open toppedvacuum channels 42, 44, a flat, gum rubber gasket 50 is positionedadjacent the upper surface of the plenum 40. The gasket 50 isrectangularly shaped and has the same approximate dimensions as theplenum 40. The gasket 50 includes a plurality of openings or apertures52 therethrough which are in fluid communication with the underlyingside vacuum channels 44. the main vacuum channel 42 is completelycovered by the gasket 50. The apertures 52 are positioned so that eachwill align with and be located immediately beneath the base 22 of onewell 18 when a culture plate 10 is positioned on the upper surface ofthe gasket 50. Therefore, the apertures 52 create individual sealed airchambers which are accessible only through the vacuum channels 42, 44.In the embodiment shown in FIGS. 4-6, the gasket 50 includes six, two bythree arrays of apertures 52. Each group of three apertures 52 islocated above a separate side vacuum channel 44. Therefore, the vacuumapparatus shown in FIGS. 4-6 will accommodate six of the culture plates10 described above and shown in FIGS. 1-3. While the vacuum apparatusshown in FIGS. 4-6 is designed for the use of six individual six-wellculture plates, it is to be understood that any number of individualplates, having a variety of number of wells, can be used by merelyproviding the vacuum channels 42, 44 and apertures 50 in the locationrequired by the particular plate or plates used.

Any vacuum induced through the vacuum hose 48 will likewise be induced,via the vacuum channels 42, 44, in each chamber formed by the aperture52 beneath the well base 22 of the culture plate 10. As the vacuum isinduced, the elastomeric membrane 26 in each well 18 will be pulleddownwardly and begin to stretch to a cured configuration. FIG. 6 showsthe downward elongation of the elastomeric membrane 26 when the vacuumis fully applied. As the vacuum is reduced, the membrane 26 returns tothe original, horizontal configuration shown in FIG. 3. The vacuumelongation of the membrane 26 may be constant or cyclic, or may beirregular or applied in a pattern as desired. Each of the six cultureplates 10 will, however, be subjected to the identical vacuumconditions.

The vacuum apparatus shown in FIGS. 4 and 5 may be subjected tocontrolled application of vacuum by a wide variety of systems. One suchsystem is shown in schematic in FIG. 7. Each vacuum apparatus, includingthe plenum 40 (not visible), gasket 50 and culture plates 10, isconnected via hose 48 to a solenoid valve 54. The solenoid valves 54 areeach connected via hose 56 to a source of vacuum 58 having a pluralityof outlets. The solenoid valves 54 are each connected via wire 60 to acomputer 62 or other control apparatus. Each solenoid valve 54 willcontrol the application of vacuum to the elastomeric membranes 26 in thewell base 22 of each culture plate 10. The computer 62 is used tocontrol the operation of the solenoid valves 54 not only to control thetiming and intensity of the vacuum supplied, but also to equilibrate thechannels 42, 44 in the vacuum plenum 40 and permit the return of ambientair as desired. Either a single solenoid valve 54 or a plurality ofvalves 54 may be used to accomplish both vacuum induction and return toambient pressure. In addition, a pressure transducer may be associatedwith each vacuum plenum, or may be positioned beneath each elastomericmembrane 26, with its output signal supplied to the computer 62. Theinformation developed by the pressure transducer may be used to controlthe actual vacuum applied.

Cells adhering to the well base 22 are subjected to commensurate stressas is applied to the well base 22 itself. The system may thus be usedfor the in vitro flexing of cell culture substrates as discussed above.The six culture plates 10 and associated vacuum apparatus may, ofcourse, be incubated in a standard incubator or may otherwise besubjected to culturing conditions as would any multi-well culture plateknown in the art.

For specific applications which do not require flexing of the cells butfor which adherence of the cells to the cell substrate is desired, asurface-modified polyorganosiloxane composition according to the presentinvention may alternatively be deposited in a six-well culture platewithout first excising the well bases. The deposited surface-modifiedpolyorganosiloxane layer may be made of any thickness, but ordinarilymembranes on the order of one millimeter are all that is required.

A variety of modifications may be made to the subject disclosure withoutchanging its nature. Six-well culture plates prepared with base anchorrings need not have base anchor ring holes 28; for example, the baseanchor ring 24 can be roughened prior to deposition of the uncuredpolyorganosiloxane so that the adherence of the siloxane resin to theactual base anchor ring surface is enhanced. The surface-modifiedpolyorganosiloxane may likewise be incorporated as the cell culturesubstrate in any number of cell culture vessels and is not limited inany way to multi-well plates. The polyorganosiloxane compositions of thepresent invention may likewise be used on the sides of culture vesselsas individual discrete particles or beads to which cells can adhere, orin any of a number of other ways that will be immediately evident to oneskilled in the art. Vacuum elongation may be induced in thepolyorganosiloxane membranes prepared as described above by any systemfor applying vacuum. Thickness of layers or membranes as prepared may becontrolled by known means to yield a substrate having predictableelongation characteristics.

Because it is believed that the present surface-modifiedpolyorganosiloxane compositions do not promote epithelial encapsulationor thickening and/or keratinization of the surrounding connectivetissues, virtually any implant or artificial organ to which celladherence is desired may be fabricated from the presentpolyorganosiloxane composition. Potential used for the surface-modifiedpolyorganosiloxanes include, but are not limited to, synthetic bloodvessels, structural prothesis including breast implants, and syntheticjoints such as knees or knuckles. For example, synthetic knuckles may befabricated from a polyorganosiloxane that is selectively aminated at theknuckle structure ends so that cells can attach to the end portions ofthe knuckle, which anchor in bone, but do not adhere to the centralknuckle area where gliding must occur. Selectively derivatizedpolyorganosiloxanes in this manner are especially suited for use asimplants because cell adherence and nonadherence may be selectivelycontrolled.

The present invention is described with greater particularity in theExamples below.

EXAMPLE 1

A 5×3-5/16 inch Falcon six-well polystyrene culture plate wasimmobilized in a wooden jig holder. The center point of the bottom ofeach well was marked. A 1-1/16 inch metal drill bit was positionedimmediately over the centered mark and the drill bit was used to drillalmost completely through the plastic. Drilling completely through wasavoided to prevent the hole from becoming oversized. The central portionof the well was pushed out using finger pressure, leaving a 4 mm. wideanchor rim at the base of the well.

The plates were inverted in the jigs and secured. Twelve evenly-spacedfrustoconical holes were drilled down into the remaining polystyrenebase of the well with a grinding bit. The plate was inverted in the jigonce again, and a stone bit was used to roughen the remainingpolystyrene ring at the base of the well as well as the side walls ofthe well. Plastic particulates were thereafter vacuumed away.

Subsequent to a 95% ethanol wash of the entire culture plate, to removecontaminants introduced by handling, the plate was inverted onto a cleansurface and the well bottoms were sealed with three inch adhesive tape.The adhesive tape was applied carefully so as to create a smooth, flatbottom to each culture plate well. The plates were inverted to anupright position.

A polyorganosiloxane composition was prepared by admixing 85 g.Dow-Corning MDX4-4210 clean grade elastomer with 15 g. of theaccompanying catalyst and 15 g. of medical grade silicone oil. Thecomponents were weighed out in a plastic disposable beaker and wereadmixed thereafter using the paint mixing bit of a drill assembly. Two60 cc. plastic syringes were filled with the resultant admixture, andthe remainder of the admixture was stored at -20° C. for later use.

Working quickly, each plate was placed on a balance and the syringeswere used to add 2.0 g. of the mixture to each well. After four plateswere poured, all four plates were placed in a centrifuge and werecentrifuged at 1,000 times gravity for 4.5 minutes (at ambienttemperature) to remove all the visually detectable air bubbles from theresin. As a result of working quickly, the resin did not becomeappreciably more viscous between dispensing and centrifugation, and themembranes in the bases of each culture plate well settled readily toform a flat surface after removal from the centrifuge.

To cure the plates, the plates were placed on a flat metal rack in anoven set at 60° C. for 45 minutes. (Had curing at elevated temperaturebeen delayed for some reason, the centrifuged plates could have beenstored at -20° C. prior to heat processing.) The plates were thenremoved from the oven and were permitted to equilibrate to roomtemperature on a flat surface for 30 minutes.

Five milliliters of 98% pur 3-aminopropyltriethoxysilane were admixed infive milliliters of 1 M. HEPES buffer, pH 7.2, with subsequent additionof deionized water to a final volume of 250 ml. Three milliliters of theresultant 3-aminopropyltriethoxysilane solution were then added to eachwell and the culture plates were covered with polyethylene film. Theplates were incubated at room temperature in the dark for 12 hours. Theresultant aminated polyorganosiloxane surfaces at the base of each cellculture well were then rinsed briefly in 2 washes of 20 mM HEPES buffer,followed by a final application of HEPES buffer, which was left in placeon the aminated surface for 15 minutes. The adhesive tape was thencarefully removed from the base of the plates; the resultant cultureplates were sterilized in ultraviolet light in a cell culture hood for12 hours, and the sterilized plates were hermetically sealed understerile conditions in a plastic overwrap.

EXAMPLE 2

The culture plates prepared in accordance with Example 1 were inoculatedand incubated in association with the vacuum apparatus illustrated inFIGS. 4-7, which cyclically elongated each well base twenty percent atthe rate of 40 times a minute. After cell culturing was complete, theoptically clear biocompatible polyorganosiloxane membrane permittedmicroscope examination of the cells without the removal of the cellsfrom the elastomeric substrate. The elastomeric substrate also provedsuitable for sampleing, and was successfully cut with each of a knife, acork borer and a trephine punch. These cut segments having cellsattached were mounted on slides, stained with fluorescent reagents andwere examined under a microscope.

EXAMPLE 3

The process according to Example 1 was repeated except that as asubstitute for centrifugation, the plates were incubated at -20° C. forfive days so that the elastomer was deaerated slowly and the resinflattened on its own. The plates were then removed from the freezer andwere cured and aminated in accordance with Example 1.

EXAMPLE 4

Biocompatible polyorganosiloxane composition well bases were prepared inaccordance with Example 1, except that amination proceeded by thefollowing method. Each cured polyorganosiloxane well base was contactedwith 1 ml. 1 N. HCl, followed by an addition of 1 ml. 1 M. NH₄ OH. Eachreagent was left in place for 30 minutes and was decanted thereafter.The plates were then washed in water, permitted to dry, and weresterilized and wrapped in accordance with Example 1.

EXAMPLE 5

Biocompatible polyorganosiloxane composition well bases were prepared inaccordance with Example 4, except that after the NH₄ OH addition andwater washing, the well bases were treated with glutaraldegyde andpeptide as follows.

One ml. 1 nanomolar glutaraldehyde was added to each well. Each well wasthen contacted with an aqueous peptide having both amine and carboxylfunctionality. Enough of the aqueous peptide was added to cover the wellbase surface. The peptide selected was 1 mM NH₂ -RGDS-COOH (R=arginine,G=glycine, D=aspartic acid and S=serine) in water. After one-half hour,the plates were washed, dried, sterilized and wrapped in accordance withExample 1.

Although the invention has been described with respect to particularembodiments and methods thereof, the invention is to be limited onlyinsofar as is set forth in the accompanying claims:

I claim:
 1. A method for treating the surface of a polyorganosiloxanecomposition comprising the steps of:(a) contacting thepolyorganosiloxane surface with an acid selected from the groupconsisting of hydrochloric acid, hydrofluoric acid and hydrobromic acidand then decanting said acid; (b) contacting the polyorganosiloxanesurface with an amine selected from the group consisting of ammoniumhydroxide, ammonium chloride and ammonium bicarbonate and then decantingsaid amine; and (c) washing said polyorganosiloxane surface with water.2. The method according to claim 1, wherein step (c) further comprisesthe step of washing said polyorganosiloxane surface with water,sequentially contacting said surface with an aldehyde and an aqueouspeptide, and washing said surface with water.
 3. The method according toclaim 1, wherein step (c) further comprises the step of washing saidpolyorganosiloxane surface with water, sequentially contacting saidsurface with glutaraldehyde and an aqueous peptide wherein said peptidehas both amine and carboxyl functionality, and washing said surface withwater.
 4. The method according to claim 1, wherein step (a) furthercomprises the step of contacting the polyorganosiloxane surface with 0.5to 1.0 ml. per cm² of 1 N. hydrochloric acide.
 5. The method accordingto claim 1, wherein step (b) further comprises the step of contactingthe polyorganosiloxane surface with 0.5 to 1 ml per cm² of 1 M. ammoniumhydroxide;.
 6. The method according to claim 1, further comprising thestep of (d) incorporating said polyorganosiloxane composition onto asolid means for supporting a cell culture to form a cell culturesubstrate.
 7. The product prepared in accordance with any one of claims1-6.
 8. A method for treating the surface of a polyorganosiloxanecomposition, comprising the co-curing of a polyorganosiloxanecomposition with an adjacent layer of a compound selected from the groupconsisting of primary amine-containing silanes, carboxyl-containingsilanes, primary amine-containing siloxanes and carboxyl-containingsiloxanes.
 9. The method according to claim 8 wherein saidpolyorganosiloxane composition is co-cured with a compound selected fromthe group consisting of 3-aminopropyltriethoxysilane,2-aminoethyitrimethoxysilane, trimethylsilylformic acid,3-(trichlorosilyl) butanoic acid, and 1,1,1-trichioro-N-(trimethylsilyl)silanamine suspended in an aqueous or aqueous buffer carrier, bymaintaining the adjacent compositions at about room temperature forabout twenty-four hours.
 10. The method according to claim 8 whereinco-curing is effected at about 60° C. and ambient pressure over a periodof about 15 minutes.
 11. The produce prepared in accordance with any oneof claims 8-10.
 12. A cell culture substrate comprising: solid means forsupporting a cell culture having at least one surface further comprisinga polyorganosiloxane composition wherein a surface of saidpolyorganosiloxane composition is treated by:(a) contacting thepolyorganosiloxane surface with an acid selected from the groupconsisting of hydrochloric acid, hydrofluoric acid, and hydrobromicacid, and then decanting said acid; (b) contacting thepolyorganosiloxane surface with an amine selected from the groupconsisting of ammonium hydroxide, ammonium chloride and ammoniumbicarbonate and then decanting said amine; and (c) washing saidpolyorganosiloxane surface with water.
 13. The cell culture substrateaccording to claim 12 wherein step (c) further comprises the step ofwashing said polyorganosiloxane surface with water, sequentiallycontacting said surface with an aldehyde and an aqueous peptide, andwashing said surface with water.
 14. The cell culture substrateaccording to claim 12, wwherein step (c) further comprises the step ofwashing said polyorganosiloxane surface with water, sequentiallycontacting said surface with glutaraldehyde and an aqueous peptidewherein said peptide has both amine and carboxyl functionality, andwashing said surface with water.
 15. The cell culture substrateaccording to claim 12 wherein step (a) further comprises the step ofcontacting the polyorganosiloxane surface with 0.5 to 1 ml. per cm² of 1N. hydrochloric acid.
 16. The cell culture substrate according to claim12 whrein step (b) further comprises the step of contacting thepolyorganosiloxane surface with 0.5 to 1 ml. per cm² of 1 M. ammoniumhydroxide.
 17. The cell culture substrate according to claim 12 furthercomprising the step of incorporating said polyorganosiloxane compositiononto a solid means for supporting a cell culture to form a cell culturesubstrate.
 18. The cell culture substrate according to claim 12 whereinsaid solid means is a multi-well polystyrene plate.